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In studying biological roles of interferon regulatory factor (IRF)-1 tumor suppressor in cervical carcinogenesis, we found that HPV E7 is functionally associated with IRF-1. Binding assays indicate a physical interaction between IRF-1 and HPV E7 in vivo and in vitro. The carboxyl-terminal transactivation domain of IRF-1 was required for the interaction. Transient co-expression of E7 significantly inhibits the IRF-1-mediated activation of IFN-beta promoter in NIH-3T3 cells. Co-transfection of E7 mutants reveals that the pRb-binding portion of E7 is necessary for the E7-mediated inactivation of IRF-1. It was next determined whether histone deacetylase (HDAC) is involved in the inactivation mechanism as recently suggested, where the carboxyl-terminal zinc finger domain of E7 associates with NURD complex containing HDAC. When trichostatin A, an inhibitor of HDAC, was treated, the repressing activity of E7 was released in a dose-dependent manner. Furthermore, the mutation of zinc finger abrogates such activity without effect on the interaction with IRF-1. These results suggest that HPV E7 interferes with the transactivation function of IRF-1 by recruiting HDAC to the promoter. The immune-promoting role of IRF-1 evokes the idea that our novel finding might be important for the elucidation of the E7-mediated immune evading mechanism that is frequently found in cervical cancer.

Transcriptional repression by sequence-specific DNA binding factors is mediated by the recruitment of a corepressor complex to the promoter region. The NK-3 homeodomain protein is a transcriptional repressor that recruits the nuclear protein kinase, homeodomain interacting protein kinase 2 (HIPK2). Here we show that HIPK2 is a component of a corepressor complex containing Groucho and a histone deacetylase complex. Groucho, like HIPK2, acts as a corepressor for NK-3 and binds to NK-3 and HIPK2. Moreover, HIPK2 appears to regulate the corepressor activity of Groucho. Transcriptional repression by NK-3 and Groucho is relieved by the histone deacetylase inhibitor trichostatin A, and both NK-3 and Groucho directly interact with the histone deacetylase HDAC1 that is associated with mSin3A in vivo. Recruitment of the histone deacetylase complex by NK-3 decreases the acetylated histones that are associated with the target gene promoter. These results indicate that NK-3 represses transcription by recruiting a complex containing Groucho and a histone deacetylase complex that leads to histone modification on chromatin and suggest that HIPK2 may play a regulatory role in the corepressor complex formation.

Two isoforms of cyclooxygenase (COX) have been identified in eukaryotic cells: a constitutively expressed COX-1 and mitogen-inducible COX-2, which is selectively expressed in response to various inflammatory stimuli. Thus, COX-2 instead of COX-1 is implicated to produce prostanoids mediating inflammatory responses. Major efforts have been focused on identifying nonsteroidal anti-inflammatory drugs (NSAIDS) which can selectively inhibit the enzyme activity of COX-2. Such NSAIDS would be more desirable anti-inflammatory agents in comparison to NSAIDS which inhibit both COX-1 and COX-2. Other than glucocorticoids, pharmacological agents which can selectively suppress the expression of COX-2 without affecting that of COX-1 have not been identified. We report here that radicicol, a fungal antibiotic, is a potent protein tyrosine kinase inhibitor, and that it inhibits the expression of COX-2 without affecting COX-1 expression in lipopolysaccharide (LPS)-stimulated macrophages with the IC50 value of 27 nM. Radicicol inhibited tyrosine phosphorylation of p53/56lyn, a Src family tyrosine kinase and one of the major tyrosine-phosphorylated proteins in LPS-stimulated macrophages. Radicicol also inhibited COX-2 expression in vivo in glomeruli of rats with experimental glomerulonephritis induced by the anti-glomerular basement membrane antibodies, in which COX-2 expression is known to be enhanced. The enzyme activity of COX-1 or COX-2 was not affected by radicicol in macrophages. Radiciciol also suppressed the COX-2 expression induced by IL-1 beta in rat smooth muscle cells. Other protein tyrosine kinase inhibitors suppressed the LPS-induced COX-2 expression in macrophages but at much higher concentrations than needed for radicicol. Radicicol did not inhibit the COX-2 expression induced by phorbol 12-myristate 13-acetate in macrophages. These results suggest that the activation of tyrosine-specific protein kinases is the proximal obligatory step in the LPS-induced signal transduction pathway leading to the induction of COX-2 expression in macrophages. The magnitude of the inhibition of COX-2 protein synthesis by radicicol was much greater than that of the steady state levels of COX-2 mRNA. These results suggest that radicicol inhibits COX-2 expression mainly at post-transcriptional steps.